XingNaoJing, prescription of traditional Chinese medicine, prevents autophagy in experimental stroke by repressing p53-DRAM pathway

Abstract

Background: Xingnaojing (XNJ), a well known prescription in traditional Chinese medicine, has been used for treatment of stroke in China. However, the effects and mechanisms of XNJ on autophagy are not clear. Here, we used the cell models of autophagy induced by serum-free condition and ischemia stroke in rats to further investigate whether the p53-DRAM pathway is involved in the effects of XNJ on autophagy.

Methods: We used the cell model of autophagy induced by serum-free condition and the rat model of ischemia caused by a middle cerebral artery occlusion (MCAO). The effects of XNJ on p53 transcriptional activity of PC12 cells were evaluated by the luciferase activity assay. The mRNA levels and the expression of p53 and its target autophagy gene DRAM (damage-regulated autophagy modulator) were analyzed respectively by Quantitative-RTPCR and Western blot assay. The activation of autophagy was detected by the levels of autophagy markers, microtubule associated protein light chain 3 (LC3) and p62 by Immunofluorescence and Western blot. p53 inhibitor was used to determine whether p53 is responsible for the effects of XNJ on preventing autophagy.

Results: The assay for luciferase activity of p53 promoter indicated that XNJ inhibited p53 transcriptional activity. XNJ reduced the expression of p53 and its target autophagy gene DRAM (damage-regulated autophagy modulator) in serum-free condition PC12 cells and the cortex in MCAO rats. XNJ reduced autophagy of PC12 cells induced by serum-free condition and the cortex in MCAO rats. Furthermore, suppression of p53 by p53 inhibitor significantly reduced the effects of XNJ on the autophagy of PC12 cells in serum-free condition.

Conclusion: XNJ prevents autophagy in experimental stroke by repressing p53/DRAM pathway. Our findings are therefore of considerable therapeutic significance and provide the novel and potential application of XNJ for the treatment of brain diseases.

Fig. 1

XNJ inhibits p53 transcriptional activity. a Serum-free condition induced the p53 transcriptional activity. PC12 cells were transfected with p53 promoter reporter construct and then stimulated with serum or serum-free condition for 6, 12, 24 and 36 h respectively. b p53 inhibitor, pifithrin α, inhibited p53 transcriptional activity. PC12 cells were transfected with p53 promoter reporter construct and then stimulated with serum or serum-free condition for 12 h in the absence or presence of pifithrin α. c XNJ inhibited p53 transcriptional activity. PC12 cells were transfected with p53 promoter reporter construct and then stimulated with serum or serum-free condition for 12 h in the absence or presence of XNJ. Luciferase activity was determined in cell lysates and normalized to Renilla activity. Results are representative of three independent experiments in duplicate

Fig. 2

XNJ inhibits p53 expression in serum-free condition PC12 cells. PC12 cells were treated with serum or serum-free condition for 48 h in the absence or presence of XNJ. a p53 mRNA was down-regulated by XNJ. The levels of p53 mRNA were analyzed by Q-PCR. b p53 expression was down-regulated by XNJ. After XNJ treatment, the cells were lysed for Western blot analysis using an enhanced chemiluminescence system. c The density of p53/β-actin band among the experimental groups was compared. Data are mean ± SEM of values obtained from three independent experiments. *, p < 0.05 compared with serum-free condition

Fig. 3

XNJ inhibits p53 target autophagy gene expression in serum-free condition PC12 cells. PC12 cells were treated with serum or serum-free condition for 48 h in the absence or presence of XNJ. a DRAM mRNA was down-regulated by XNJ. The levels of DRAM mRNA were analyzed by QRT-PCR. b DRAM expression was down-regulated by XNJ. After XNJ treatment, the cells were lysed for Western blot analysis using an enhanced chemiluminescence system. c The density of DRAM/β-actin band among the experimental groups was compared. Data are mean ± SEM of values obtained from three independent experiments. *, p < 0.05 compared with serum-free condition

Fig. 4

XNJ reduced autophagy of PC12 cells induced by serum-free condition. PC12 cells were treated with serum or serum-free condition for 48 h in the absence or presence of XNJ. a The LC3-positive cells were analyzed using immunofluorescence (upper panel, original magnification, 200×), and comparison of the percentage of LC3-positive cells among the experimental groups was showed (lower panel). b The expression levels of LC3 and p62 were analyzed using Western blot assay (left panel). The density of LC3-II and p62/β-actin band was compared among the experimental groups (right panel). Data are mean ± SEM of values obtained from three independent experiments. *, p < 0.05 compared with serum-free condition

Fig. 5

p53 is responsible for the effects of XNJ on preventing autophagy. a Suppression of p53 by p53 inhibitor significantly reduced the effects of XNJ on the autophagy. PC12 cells were cultured in serum-free condition and stimulated with p53 inhibitor in the absence or presence of XNJ, the expression levels of LC3 and p62 were analyzed using Western blot assay. b Comparison of the density of LC3-II/β-actin band among the experimental groups was showed. c Comparison of the density of p62/β-actin band among the experimental groups was exhibited. *, p < 0.05 compared with the serum-free condition group

Fig. 6

XNJ attenuated autophagy induced by cerebral ischemia. a The LC3-positive cells were analyzed using immunofluorescence 1 d after MCAO (upper panel, original magnification, 200×), and comparison of the percentage of LC3 positive cells among the experimental groups was exhibited (lower panel). Data are mean ± SEM of values obtained from 6 brains. b The levels of LC3 and p62 were analyzed by Western blot analysis. c Comparison of the density of LC3-II, p62/β-actin band among the experimental groups was showed. Data are the mean ± SEM of values obtained from 6 brains. *, p < 0.05, compared with the group of MCAO rats treated with vehicle

Fig. 7

XNJ inhibited p53 and DRAM expression induced by cerebral ischemia. a Levels of p53 mRNA in the ipsilateral cortex were analyzed by real-time RT-PCR. b Levels of p53 and DRAM in the ipsilateral cortex were analyzed by Western blot. c The density of p53 and DRAM/β-actin band among the experimental groups was compared. Data are the mean ± SEM of values obtained from 6 brains. *, p < 0.05, compared with MCAO rats treated with vehicle